The regulation of cyclic 3',5'-nucleotide phosphodiesterase will be investigated utilizing a Ca 2ion-dependent phosphodiesterase from brain as a model system. In the presence of low concentrations of calcium, this phosphodiesterase is activated by a homogeneous Ca2 ion-binding protein isolated from brain. The extent to which the phosphodiesterase activity depends on interaction with the Ca2 ion-binding protein will be studied by gel filtration chromatography, sucrose density gradient centrifuation, electrophoresis, and analyses of reaction kinetics. The sensitive phosphodiesterase will be purified from pig brain and characterized physically and kinetically. Direct Ca2 ion binding studies and Ca2 ion analyses will be conducted with enzyme from which the protein activator has been removed to determine whether or not the enzyme per se has binding sites for Ca2 ion. Tissue surveys and subcellular fractionations will be conducted for both the activator and the phosphodiesterase to gain initial insight into the physiological significance of the Ca2 ion dependence. Later, attempts will be made to identify a role for the Ca2 ion-dependent phosphodiesterase in neural transmission. The enzyme will be sought in isolated synaptosomes and in tissue culture systems of astrocytoma (C-6) and neuroblastoma (C-1300). The Ca2 ion-dependent phosphodiesterase will be investigated for possible physical and kinetic relationships with other forms of cyclic nucleotide phosphodiesterase. In particular, it will be compared to a Ca2 ion-inhibited cyclic nucleotide phosphodiesterase of smooth muscle. The possibility that other cyclic nucleotide phosphodiesterases are regulated by analogous small molecule-binding protein-catalytic protein interactions will be explored.